Immunoglobulins, also known as antibodies, are the major secretory products of the immune system. They are typically formed of basic structural units—each with 2 heavy chains and 2 light chains—to form monomers with one such unit, dimers with two units, pentamers with five units, or hexamers with six units. Antibodies play a significant role in innate immunity. In a natural immune response to a pathogen, complexes are formed between the pathogen and antibodies. These immune complexes activate a wide range of effector functions, thus leading to the killing, removal and destruction of the pathogen. Antibodies can also react with the body's own antigens, which can lead to autoimmune diseases, and contribute to chronic inflammatory symptoms. Antibodies can have an anti-inflammatory activity, for example by targeting and neutralising various mediators in the inflammatory cascade.
There are five major isotypes of immunoglobulins, which perform different roles. IgG provides the majority of antibody-based immunity against invading pathogens, and is discussed in more detail below. IgM occurs in a membrane-bound form and in solution. In solution, it typically forms a pentamer, providing high avidity in binding to the antigen. It is often the first, immediate defence against infections before sufficient specific IgG is produced. IgA usually occurs as a dimer and is found in mucosal areas, e.g. the gut, lung and urogenital tract. It protects these surfaces against colonization by pathogens. IgE is mainly involved in allergic reactions. It binds to allergens and triggers histamine release from mast cells and basophils. IgD is found mainly on the surface of B lymphocytes that have not been exposed to antigens.
In humans, four subclasses of IgGs are defined and numbered according to their relative concentrations in normal serum: IgG1, IgG2, IgG3 and IgG4, which respectively account for approximately 60%, 25%, 10% and 5% of serum IgG, each IgG subclass possessing unique effector functions.
In general, each individual monomeric immunoglobulin unit, e.g. an IgG molecule, consists of two identical light chains and two identical heavy chains, which in turn comprise repeating structural motifs of approximately 110 amino acid residues. Domains of the light and heavy chains pair in covalent and non-covalent associations, thus forming three independent protein moieties connected through a flexible linker, the so-called hinge region. Two of these moieties are referred to as Fab (antigen-binding fragment) regions and are of identical structure. Each of the Fab regions forms the same specific antigen-binding site. The third moiety is the Fc (crystallizable fragment) region, which forms interaction sites for ligands that activate clearance and transport mechanisms.
IgGs play an important role in diseases. IgG1-type antibodies are the most commonly used antibodies in cancer immunotherapy where antibody-dependent cell-mediated cytotoxicity (ADCC) is often deemed important. Furthermore, IgGs are known to mediate both pro- and anti-inflammatory activities through interactions mediated by their Fc fragments. On one hand, interactions between Fc and its respective receptors are responsible for the pro-inflammatory properties of immune complexes and cytotoxic antibodies. On the other hand, intravenous gamma globulin (IVIG) and its Fc fragments are anti-inflammatory and are widely used to suppress inflammatory diseases. The precise mechanism of such paradoxical properties is unclear but it has been proposed that glycosylation of IgG is crucial for regulation of cytotoxicity and inflammatory potential of IgG.
To date, research into the role of glycosylation in the regulation of cytotoxicity and the inflammatory effects mediated by IgGs has mainly focused on the Fc region. Glycosylation of IgG is essential for binding to all FcyRs by maintaining an open conformation of the two heavy chains. This requirement of IgG glycosylation for FcyR binding explains the inability of de-glycosylated IgG antibodies to mediate in vivo triggered inflammatory responses, such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis and the release of inflammatory mediators. A link between autoimmune states and specific glycosylation patterns of IgG antibodies has been observed in patients with rheumatoid arthritis and several autoimmune vasculities in which decreased galactosylation and sialylation of IgG antibodies have been reported (Parekh et al., Nature 316, 452 (1985); Rademacher et al., Proc. Natl. Acad. Sci. USA 91, 6123 (1994); Matsumoto et al., 128, 621 (2000)).
International patent application WO 2007/117505 discloses a polypeptide containing at least one IgG Fc region, said polypeptide having a higher anti-inflammatory activity and a lower cytotoxic activity as compared to an unpurified antibody. In this application it was found that an IgG preparation enriched for increased sialylation of the N-linked glycosylation site on the Fc fragment showed an enhanced anti-inflammatory activity in vivo. In contrast, it was concluded that Fab fragments displayed no anti-inflammatory activity in this in vivo assay.
The international application WO 2008/057634 by the same inventors as WO 2007/117505 discloses a polypeptide containing at least one IgG Fc region, wherein said at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a α-2,6 linkage. The polypeptide of WO 2008/057634 has a higher anti-inflammatory activity as compared to an unpurified antibody. Also in this application it was found that an IgG preparation enriched for increased sialylation of the N-linked glycosylation site on the Fc fragment showed an enhanced anti-inflammatory activity in vivo. In contrast, it was concluded that Fab fragments displayed no anti-inflammatory activity in this in vivo assay. Further evidence that the anti-inflammatory activity is due to increased sialylation on the Fc fragment, rather than Fab fragment, is provided in international application WO 2009/079382 by the same inventors.
In a commentary by Nimmerjahn & Ravetch (JEM 204, 11-15 (2007), the authors clearly teach that the sialylation in the Fc region is the key to the anti-inflammatory activity of IVIG. In addition they state that a generalized role for the antigen binding domain, located in the Fab regions, in the anti-inflammatory activity of IgG is unlikely, given that in their hands, intact IVIG and its Fc fragments have equivalent anti-inflammatory activity.
The international application WO 2007/005786 is directed to methods for controlling the properties of an Fc-containing molecule, said methods comprising altering the sialylation of the oligosaccharides in the Fc region. In this application it was found that the level of sialylation of the Fc oligosaccharides alters the affinity of recombinantly-produced therapeutic antibodies for Fcγ receptors, resulting in modulation of various aspects of the biological actions of these antibodies. It was further discovered that the removal of sialic acid from the Fc oligosaccharides enhances the avidity of recombinantly-produced therapeutic antibodies for their target molecule. However, the effect is believed to be entirely Fc mediated, as no differences in the intrinsic affinity between each Fab arm and the target were observed. The inventors of WO 2007/005786 hypothesize that the removal of the charged static group from the Fc oligosaccharide allows for more flexibility in the overall antibody structure, thus providing a higher potential of interaction for the two binding domains in relationship of one to the other.
A recent review article by Jefferis (Jefferis, R.; Nat Rev Drug Discov. 2009 March; 8(3):226-34) discusses glycosylation as a strategy to improve antibody-based therapeutics. According to Jefferis 2009, approx. 30% of polyclonal human IgG molecules bear N-linked oligosaccharides in the IgG Fab region and a higher level of galactosylation and sialylation for IgG Fab than for IgG Fc is observed. When present, the glycosylation is attached to the variable regions, and the functional significance of IgG Fab glycosylation of polyclonal IgG is not clear. Based on studies on monoclonal antibodies it is speculated that the glycosylation in the Fab region of an antibody can have a neutral, positive or negative influence on antigen binding. No incentive is given to study this further, nor to enrich the antibody population that is glycosylated in the Fab region.
Thus, antibody preparations having an increased amount of sialylation in the Fc region of antibodies have been intensely studied for the prevention and treatment of various diseases and also a potential role of glycosylation of the Fab region of antibodies has been discussed. However, whereas a number of antibodies are currently in use, there is still a need to provide alternative and/or improved antibody preparations having a more beneficial effectiveness in clinical applications.
The solution to this technical problem is achieved by providing the embodiments characterised in the claims. The inventors have surprisingly shown that enrichment of antibodies that show altered sialylation in the Fab region have altered immunomodulatory properties.